Apoptosis in NK Cells. Natural killer (NK) cells exhibited ligand- induced death similar to activated T cells, when primed by interleukin 2 (IL-2), IL-12 and IL-15 and triggered by occupancy of the CD16 receptor. We have examined the mechanisms involved in NK apoptosis and compared these cells to the reported studies performed in T cells. Selected genes have been reported as critical for T-cell apoptosis, such as Nur77, c-myc, and BCL2; however, these appeared not to be regulated and required during NK cell apoptosis. Metabolic [Act. D, Wortmannin] and transport [Nac, CsA, FK506, MgEGTA] inhibitors were examined for interference with the CD16-induced apoptosis, with only Nac inhibiting cell death. ICE proteases, shown important for Fas-induced T-cell apoptosis, were examined for their effect on NK lysis and apoptosis. CD16-induced apoptosis was not blocked by ZVAD, whereas Fas and Staursporin induced death was blocked by this ICE inhibitory peptide. By contrast, CD16-induced death was potently blocked by a cathepsin B inhibitor, ZFA. Therefore, NK cells undergo rapid apoptotic episode when CD16 is cross-linked. However, the Fas receptor pathway in NK and T cells has similar requirements. NK target cellular lysis, mediated by the exocytosis of perforin and granzymes, was not blocked by either ZVAD or ZVA peptides. These results suggest a complex balance of pathways that can be used by NK cells to kill targets and to be regulated via apoptosis by the immune system. Monocyte Regulation. The antineoplastic agent bryostatin-1 (bryo-1) can potently induce the production of pro- inflammatory cytokines from human peripheral blood monocytes. Stimulation of monocytes increased levels of IL-8 mRNA and induced the expression of IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL- 6 mRNA and the secretion of all four proinflammatory cytokines. Bryo-1 synergized with IL-2 in part, based on the ability of bryo-1 to upregulate IL-2Rgamma chain expression. The effects of bryo-1 could be blocked by the protein kinase C (PKC) inhibitors. These results show for the first time that bryo-1 is a powerful activator of human monocytes and suggest that stimulation of monokine secretion by bryo-1 may represent at least one of the mechanisms responsible for the in vivo antitumor activity of this drug. We have previously reported that a 19- base pair (bp) element of the 5'-flanking region of the iNOS gene containing a sequence homology to a hypoxia-responsive enhancer (iNOS- HRE), mediates hypoxia-induced activation of the iNOS promoter in IFN- gamma-treated murine macrophages. The iron chelator desferrioxamine (DFX) was examined for regulation of the iNOS promoter and iNOS gene expression. DFX induced DNA-binding activity to the hypoxia-inducible factor 1 (HIF-1) consensus sequence of the iNOS promoter and activated the iNOS-HRE. Functional analysis of the 5'-flanking region of the iNOS gene demonstrated that IFN-gamma plus DFX activated the full-length iNOS promoter and that the iNOS-HRE was required for DFX-induced iNOS transcriptional activity. These data establish that DFX is a costimulus for the transcriptional activation of the iNOS gene in IFN-gamma-treated macrophages and they provide evidence that the iNOS-HRE is required for the DFX-dependent activation of the iNOS promoter. Furthermore, our results indicate that the iNOS-HRE is a regulatory element of the iNOS promoter responsive to iron chelation.